Glycated proteins are generated by non-enzymatic covalent bonding between aldehyde groups in aldoses, such as glucose (monosaccharides potentially containing aldehyde groups and derivatives thereof), and amino groups in proteins, followed by Amadori rearrangement. Examples of amino groups in proteins include α-amino groups of the amino terminus and side chain ε-amino groups of the lysine residue in proteins. Examples of known glycated proteins generated in vivo include glycated hemoglobin resulting from glycation of hemoglobin and glycated albumin resulting from glycation of albumin in the blood.
Among such glycated proteins generated in vivo, hemoglobin Ale (HbAlc) has drawn attention as a glycemic control marker significant for diagnosis of diabetic patients and control of conditions in the field of clinical diagnosis of diabetes mellitus. HbAlc is a protein comprising glucose bound to the α-amino group at the N-terminal (amino-terminal) valine (Val) residue of the hemoglobin “β chain.” The blood HbAlc level reflects the average blood glucose level for a given period of time in the past, and the measured value thereof serves as a significant indicator for diagnosis and control of diabetes conditions.
Several types of enzymatic methods involving the use of amadoriases have heretofore been known as methods for rapidly and simply measuring HbAlc.
Enzymes that oxidize iminodiacetic acid or a derivative thereof (also referred to as an “Amadori compound”) in the presence of oxygen to catalyze a reaction to generate glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide are collectively referred to as “amadoriases.” Amadoriases are known to be useful for measuring HbAlc by an enzymatic method. Amadoriases have been found in bacteria, yeast, and fungi. For example, amadoriases derived from the genera Coniochaeta. Eupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Aspergillus, Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella, Achaetomium, Thielavia, Chaetomium, Gelasinospora, Microascus, Leptosphaeria, Ophiobolus, Pleospora, Coniochaetidium, Pichia, Debaryomyces, Corynebacterium, Agrobacterium, and Arthrobacter have been reported (e.g., Patent Documents 1 and 6 to 15 and Non-Patent Documents 1 to 9). These genera may be referred to as the genera Coniochaeta etc. in this description. In some of the aforementioned documents, amadoriase is occasionally referred to as, for example, ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, or fructosyl amine oxidase.
As a method for rapidly and readily measuring HbAlc with the use of various types of amadoriases as described above, a method in which HbAlc is degraded with a cleavage enzyme such as a protease and a particular target substance released from the β-chain amino terminus of HbAlc is quantified with the use of amadoriases as described above is known (e.g., Patent Documents 1 to 7).
Specifically, a method in which HbAlc is degraded with a particular protease or the like, α-fructosyl valyl histidine (hereafter referred to as “αFVH”) is released from the β-chain amino terminus thereof, and the released αFVH is quantified has been known. At present, such method is a major technique for measuring HbAlc by an enzymatic method.
According to another method that has been proposed as a method for quantifying HbAlc, α-fructosyl valine (hereafter referred to as “αFV”) is released and the released αFV is quantified. According to this method, however, various contaminants are disadvantageously cleaved from various sites other than the N-terminus of the target HbAlc hemoglobin “β-chain” in the process of cleavage of αFV from HbAlc. Thus, such method is problematic in terms of accuracy.
When implementing such methods for measuring HbAlc, it is important to select, search for, or create amadoriases exhibiting the maximal reaction specificity to a particular target substance in order to perform accurate measurement. Such attempts are been continuously made to this day.
According to a third method for measuring HbAlc by an enzymatic method involving the use of amadoriases, α-fructosyl hexapeptide comprising 6 amino acids including valine at the glycated β-chain amino terminus (α-fructosyl-valyl-histidyl-leucyl-threonyl-propyl-glutamic acid, hereafter referred to as “αF6P”) is released and then quantified (e.g., Patent Documents 17 and 18).
According to a conventional technique for measuring HbAlc defined by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), the HbAlc β-chain is digested with the use of a Glu-C protease to release αF6P, and the released αF6P is measured via HPLC-CE (HPLC capillary electrophoresis) or HPLC-MS (HPLC-mass analysis) to determine the HbAlc level (Non-Patent Document 10).
Because a special apparatus and complicated procedures at the time of detection is needed for this method, development of an enzymatic method had been desired. However, the aforementioned method defined by IFCC has heretofore been widely recognized as a reference method excellent in specificity to this day and before an enzymatic method of HbAlc measurement became widespread. Accordingly, such technique has been employed in laboratory institutions at which equipment and techniques necessary for such method are available.
When comparing the major enzymatic method comprising measuring αFVH using amadoriases which is the mainstream method for measuring HbAlc at this day with the reference method for the measurement of HbAlc via HPLC-CE or HPLC-MS defined by IFCC, the former method focuses on α-fructosyl dipeptide; that is, αFVH, as the target of measurement, whereas, the latter method focuses on α-fructosyl hexapeptide; that is, αF6P, as the target of measurement. If a method of enzymatic measurement based on the same principle as the reference method in which HbAlc is measured through quantification of αF6P is realized, the relevance between the reference method and the method of enzymatic measurement can be enhanced. Accordingly, such technique has a great significance at the industrial level. However, most of known amadoriases show reactivity selectively to relatively short α-fructosyl peptides, and, unfortunately, practical amadoriases that can satisfactorily react with αF6P released from the β-chain amino terminus of HbAlc and rapidly quantify the released αF6P (hereafter referred to as “fructosyl hexapeptide oxidase” (F6P oxidase)) have not yet been discovered.
According to the IFCC reference method, a cleavage enzyme, i.e., Glu-C protease, is applied to HbAlc to cleave αF6P from the β-chain amino terminus of HbAlc (Non-Patent Document 10). However, neither α-fructosyl amino acid nor α-fructosyl dipeptide is substantially cleaved even if Glu-C protease is allowed to react with αF6P (Patent Document 16). Accordingly, substantially all of glycated peptides cleaved from the β-chain N terminus glycated upon cleavage of HbAlc with Glu-C protease remain in the form of αF6P, and substantially no peptides are degraded to peptides of shorter chains. Most known amadoriases show high-level reactivity selectively with relatively short α-fructosyl peptides, and accordingly it is not possible to quantify αF6P.
Examples of amadoriases capable of reacting with αF6P that have been disclosed include amadoriases derived from plants belonging to the family Zingiberaceae (Patent Document 17), amadoriases derived from Aspergillus oryzae (Patent Document 18), and amadoriases derived from Phaeosphaeria nodorum (Patent Documents 15 and 19).
Fructosyl peptide oxidase derived from plants belonging to the family Zingiberaceae was reported as the first amadoriase reacting with αF6P (Patent Document 17). However, it would take as long as about 16 hours in order to detect αF6P with the use of a crude enzyme solution. Thus, this can hardly serve as a practical αF6P oxidase. Since this enzyme is unpurified and derived from a plant, also, it is predicted that various technical problems would arise when such enzyme is applied to a general procedure of enzyme mass-production utilizing genetically engineered microorganisms. In fact, there is no report that such enzyme is purified, mass-produced, or put to practical applications.
As a different approach, it has been reported that a plurality of amadoriases derived from the genus Aspergillus are capable of reacting with αF6P (Patent Document 18). However, it takes as long as 4 hours to conduct a reaction so as to detect αF6P oxidation activity in a crude enzyme solution. Accordingly, it is deduced that the productivity of αF6P oxidases derived from the genus Aspergillus is very low or reactivity per amadoriase molecule; i.e., specific activity, is very low. Low productivity is not favorable from the viewpoint of industrial applicability. In addition, a lower specific activity would necessitate the use of a larger amount of enzyme when preparing a composition or kit for αF6P measurement.
In addition to amadoriases derived from the genus Aspergillus, amadoriases derived from Phaeosphaeria nodorum were found to be reactive with αF6P (Patent Document 15). However, practicality of such enzyme is still at the developmental stage, and its specific activity is as low as 0.0013 U/mg. This specific activity is less than 1/18,300 of the specific activity of amadoriase derived from the genus Coniochaeta with αFVH (i.e., 23.8 U/mg) that are commonly used for a method, which is a currently major enzymatic measurement technique, in which αFVH is released from HbAlc and quantified (Non-Patent Document 1). This necessitates the use of considerably large amounts of enzymes so as to measure αF6P with the use of amadoriases derived from Phaeosphaeria nodorum, and accordingly, use of such enzyme is not realistic.